Abstract:This article used special medical formula milk powder as the raw material to optimize and determine the best method for detecting five nucleotides in this product by using ultra-high-performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）.By optimizing the pre-processing methods and instrument conditions，the final pre-processing method was adopted under the following conditions：0.80 g of sample was taken，and the solvent temperature was 40 ℃.40 mL of solvent was used，with a pH regulator of 25% acetic acid solution to adjust the final pH to 4.2±0.1.The UPLC-MS/MS and detection conditions were optimized as follows：electrospray ionization and ion mode and multiple response monitoring scans were used，and 5 mmol/L ammonium acetate-0.1% formic acid solution and acetonitrile was used as mobile phase for gradient elution.Under these conditions，the average recovery rates for the five nucleotides ranged from 91.865% to 100.815%.This method could rapidly and accurately determine the content of the five nucleotides in special medical formula milk powder.